Viruses of the family Paramyxoviridae have negative-sense single-stranded RNA as the genome. Their vectors capable of highly expressing a foreign gene have been prepared by reverse genetics methods and considered to have excellent properties as vectors for gene therapy or vectors for recombinant vaccines. The life cycles of these viruses are free from DNA phase. In addition, all processes of transcription and replication occur cytoplasmically outside the nucleus. For these reasons, the probability of homologous recombination with chromosomal DNA is very small, and the risk of developing cancer due to gene recombination into the chromosome is also small.
The present inventors have previously attempted to vectorize human parainfluenza virus type 2 (hereinafter, referred to as hPIV2), a negative-sense single-stranded RNA virus, belonging to the genus Rubulavirus within the subfamily Paramyxovirinae of the family Paramyxoviridae, and studied use thereof as vectors for vaccines and for gene therapy.
In this process, the present inventors have constructed an F gene-defective vector of hPIV2 completely lacking the F gene of a viral structural protein. This vector has non-transmissible or single-round-infectious properties. The present inventors have also successfully obtained stably F gene-expressing packaging cells having the ability to produce high-titer vectors for production of the F gene-defective vector. The F gene-defective vector can yield an infectious vector carrying the F protein on the virus envelope, only in packaging cells expressing F gene. On the other hand, in cells or tissues expression of F gene, this vector can do so-called self-replication, but cannot yield an infectious vector. Inexhaustible vector multiplication therefore does not occur in recipients. Thus, this vector is highly safe (Patent Literature 1).
Nonclinical trials, clinical research, clinical trials, etc., have reported many results indicating the efficacy of vaccines or gene therapy using recombinant live RNA virus vectors or attenuated recombinant RNA viruses (Non Patent Literatures 1 to 9). At the moment, however, there is no report on recombinant live RNA virus vector preparations approved as medicines by the Food and Drug Administration and the European Medicines Agency. This is presumably because in the case of using recombinant live RNA viruses as vectors, large amounts of viruses and foreign gene products are still produced in infected cells, though safety measures are taken in such a way that pathogenicity is reduced by use of attenuated viruses or non-transmissible vectors are utilized; thus concerns such as their influence on homeostasis or mutations of the foreign genes cannot be dispelled.
Thus, vaccines whose genome has been inactivated or degraded into components so as to prevent the transcription or replication of the virus are highly safe. Treatment methods with formalin are often used in the preparation of such inactivated vaccines.
Unfortunately, the inactivated vaccines, which are considered to be highly safe, also present safety problems. In the 1960s, a formalin-inactivated RSV vaccine was developed from RSV belonging to the family Paramyxoviridae, and vaccination with this vaccine resulted in severe infant patients and two cases of death after the natural infection of RSV. Then, the cause thereof was intensively investigated. The inactivation treatment of the virus with formalin was found to alter the three-dimensional structure of F membrane protein. It was revealed that antibodies having no neutralizing activity against the F membrane protein altered by the vaccination are produced, and the antibody having neutralizing activity against the F membrane protein having a normal structure is not produced because the F membrane protein having a normal structure is absent due to the formalin treatment. After the vaccination, the natural infection of the RSV virus causes the response of the body to excessively produce the antibodies having no neutralizing activity in order to suppress the multiplication of the virus. As a result, a large number of eosinophils, which are not seen in common RSV infection, are infiltrated into lung tissues. Along with this, the levels of cytokines such as IL-4 and IL-5 are elevated so that neither local IgA antibodies nor cellular immunity is induced, probably leading to increased severity.
In addition, the formalin-inactivated RSV is known to be inferior in the ability to maturate dendritic cells, which are antigen-presenting cells, to live RSV. Inactivation by UV treatment, heat treatment, or the like, without the use of formalin has also been studied, but has not yet been put in practical use because all of these treatments have the low ability to activate dendritic cells and further induce inflammatory response. Likewise, the infiltration of eosinophils and atypical measles have also been reported as to a formalin-inactivated vaccine of measles virus belonging to the family Paramyxoviridae.
Meanwhile, animal testing has reported that an inactivated RSV vaccine designed to be able to induce Th1 immunity is effective for preventing viral infection. This suggests the possibility that an inactivated vaccine that can induce Th1 can solve these problems.
Virus vectors are considered to offer high in vivo transgene expression. Some reports, however, show that these vectors are not highly workable as vectors for gene therapy, because the transgene expression in virus-infected cells or tissues is not quantitative or has a low expression level. The suppression of expression by pre-existing antibodies against the transferred virus vectors or the suppression of expression by antibodies against the virus vectors associated with a plurality of doses of the vectors has also been reported. These reports imply the difficulty in using virus vectors having the high ability to induce antibodies, in gene therapy, which requires over a certain level of gene expression, albeit transient.
In the case of transferring a foreign gene to a virus vector and expressing the gene, the foreign gene product has no packaging signal and, therefore, is usually not contained in the vector. The virus vector after in vivo administration infects a recipient cell where transcription and replication in turn occur so that the foreign gene product is produced for the first time. Thus, the expression of the foreign gene is suppressed or reduced when neutralizing antibodies or the like inhibit the infection, transcription or replication, etc., of the virus vector.
The efficiency of viral infection of cells or tissues is low in vivo, or even if the cells or tissues are virus-infected, transcription or replication does not occur or occurs with very poor efficiency in vivo so that the foreign gene is hardly expressed in most cases. This is probably because, for example: in in vivo infected cells, interferons or the like induced by the viral infection minimize the transcription or replication of the virus; and extracellular matrix or the like hinders the virus vector from reaching a target cell.